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. 2018 Oct;17(10):2206-2216.
doi: 10.1158/1535-7163.MCT-17-0836. Epub 2018 Jul 3.

Inhibition of Parp1 by BMN673 Effectively Sensitizes Cells to Radiotherapy by Upsetting the Balance of Repair Pathways Processing DNA Double-Strand Breaks

Aashish Soni  1 , Fanghua Li  1 , You Wang  1 , Martha Grabos  1 , Lisa Marie Krieger  1 , Shipra Chaudhary  1 , Mohammad Sharif Mortoga Hasan  1 , Mansoor Ahmed  2 , C Norman Coleman  2 , Beverly A Teicher  3 , Richard L Piekarz  4 , Dian Wang  5 , George E Iliakis  6
Affiliations

Inhibition of Parp1 by BMN673 Effectively Sensitizes Cells to Radiotherapy by Upsetting the Balance of Repair Pathways Processing DNA Double-Strand Breaks

Aashish Soni et al. Mol Cancer Ther. 2018 Oct.

Abstract

Parp inhibitors (Parpi) are commonly used as single agents for the management of tumors with homologous recombination repair (HRR) deficiencies, but combination with radiotherapy (RT) is not widely considered due to the modest radiosensitization typically observed. BMN673 is one of the most recently developed Parpi and has been shown to mediate strong cell sensitization to methylating agents. Here, we explore the mechanisms of BMN673 radiosensitization to killing, aiming to combine it with RT. We demonstrate markedly stronger radiosensitization by BMN673 at concentrations substantially lower (50 nmol/L) than olaparib (3 μmol/L) or AG14361 (0.4 μmol/L) and dramatically lower as compared with second-generation inhibitors such as PJ34 (5 μmol/L). Notably, BMN673 radiosensitization peaks after surprisingly short contact times (∼1 hour) and at pharmacologically achievable concentrations in vivo BMN673 exerts a complex set of effects on DNA double-strand break (DSB) processing, including inhibition of classic nonhomologous end-joining (cNHEJ) and alternative end-joining (altEJ) pathway at high doses of ionizing radiation (IR). BMN673 enhances resection at DSB and favors HRR and altEJ at low clinically relevant IR doses. The combined outcome of these effects is an abrogation in the inherent balance of DSB processing culminating in the formation of chromosomal translocations that underpin radiosensitization. Our observations pave the way to clinical trials exploring inherent benefits in combining BMN673 with RT for the treatment of various forms of cancer. Mol Cancer Ther; 17(10); 2206-16. ©2018 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1.
Figure 1.
Among Parpi, BMN673 exerts the strongest radiosensitization. Exponentially growing cells were treated with indicated inhibitors for 1 hour, irradiated, trypsinized, and seeded in appropriate numbers in the presence of inhibitors to form colonies for 7 to 8 days. Drug toxicity is indicated by reduced surviving fraction at 0 Gy. A, Impact of various Parpi on radiosensitivity in CHO cells. B, Comparison of clonogenic survival of BMN673-treated CHO cells with Xrs6 (Ku80m) or irs1SF (Xrcc3m) cells. C, Effect of various concentrations of BMN673 on CHO radiosensitization. D, Impact of Parp2 and Parp3 inhibition on CHO radiosensitization. Data, mean ± SD calculated from three to four independent experiments.
Figure 2.
Figure 2.
BMN673 efficiently radiosensitizes human rhabdoid and sarcoma cell lines. Exponentially growing cells were treated with indicated inhibitors for 1 hour, irradiated, trypsinized, and seeded to form colonies either under drug-free conditions or under continuous exposure to inhibitors. Drug toxicity is indicated by the surviving fraction at 0 Gy. A, Impact of short (1 hour) vs. continuous treatment with BMN673 on CHO radiosensitization. B, Impact of short (1 hour) vs. continuous treatment with 50 nmol/L BMN673 on radiosensitization in BT12 rhabdoid human cells. C, Effect of PJ34 vs. BMN673 continuous treatment on radiosensitization in BT12 rhabdoid human cells. D, Impact of short (1 hour) vs. continuous pretreatment with 10 nmol/L BMN673 on radiosensitization in CHLA9 Ewing sarcoma cells. Data, mean ± SD calculated from three to four independent experiments.
Figure 3.
Figure 3.
BMN673 radiosensitization is specific for tumor cells. Exponentially growing cells were treated with indicated inhibitors for 1 hour prior to IR (continuous treatment was toxic in these cell lines), immediately trypsinized, seeded at appropriate numbers in the absence of BMN673, and allowed to form colonies for 7 to 8 days in drug-free medium. A, Impact of short (1 hour) pretreatment with BMN673 on radiosensitization in U2OS cells. B, As in A for HCT116 cells. C, As in A for A549 cells. D, As in A for normal human fibroblast 82-6 hTert cells. E, As in A for normal RPE-1 cells. F, BMN673 radiosensitization after fractionated irradiation (3 fractions of 2 Gy separated by 4 or 24 hours) in RPE-1 cells. G, As in F for A549 cells. H, As in F for HCT116 cells. The significance of differences between individual measurements is indicated by connecting lines between bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant. Data, mean ± SD calculated from three independent experiments.
Figure 4.
Figure 4.
BMN673 promotes resection and increases Rad51 foci formation. Cells were exposed to 2 Gy IR in the presence or absence of BMN673 and kinetics of RPA70, 53BP1, and Rad51 foci formation, and decay was measured by immunofluorescence. A, Representative images showing formation and resolution of RPA70 foci after 2 Gy in the absence or presence of 50 nmol/L BMN673. B, Graphical representation of RPA70 foci formation and resolution without background (0 Gy) correction. C, Graphical representation of RPA70 foci formation and resolution after background correction. D, Representative images showing formation and resolution of 53BP1 foci after 2 Gy in the absence or presence of 50 nmol/L BMN673. E, Graphical representation of 53BP1 foci formation and resolution without background correction. F, Graphical representation of 53BP1 foci formation and resolution after background correction. G, Representative images showing formation and resolution of Rad51 foci after 2 Gy in the absence or presence of 50 nmol/L BMN673. H, Graphical representation of Rad51 foci formation and resolution at 1, 3, and 8 hours after IR. Data, mean ± SD calculated from two to three independent experiments. The significance of differences between individual measurements is indicated by connecting lines between bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant.
Figure 5.
Figure 5.
BMN673 treatment causes translocations in irradiated cells. Cells were exposed to 1 Gy X-rays in the presence or absence of the indicated inhibitors and analyzed for chromosome damage at metaphase 4 hours after IR to limit analysis to cells irradiated in G2 phase of the cell cycle. The protocol is a modification of one previously used for this type of analysis. Chromatid translocations were scored. No translocations were detected in unirradiated cells. A, Representative image of IR-induced chromatid translocation (indicated by arrow). B, Translocations forming in CHO cells after exposure to 1 Gy IR and treatment with NU7441 and various Parpi as indicated. C, Translocations in HCT116 WT and RPE-1 cells after exposure to 1 Gy, alone or in combination with 50 nmol/L BMN673. Data, mean ± SD calculated from two to three independent experiments. The significance of differences between individual measurements is indicated by connecting lines between bars; *, P < 0.05; n.s., nonsignificant.
Figure 6.
Figure 6.
At high doses of IR, BMN673 compromises cNHEJ and altEJ. Exponentially growing cells were irradiated with 20 Gy in the presence or absence of indicated inhibitors. A total of 500 nmol/L BMN673, 3 μmol/L olaparib, 5 μmol/L PJ34, and 5 μmol/L NU7441 were added 1 hour prior to IR and maintained during the experiment. Repair kinetics were measured using PFGE. A, DSB repair kinetics in CHO cells incubated in the presence or absence of indicated inhibitors. B, DSB repair kinetics in MEFs incubated with the indicated Parpi. C, DSB repair kinetics measured in Parp1−/− MEFs incubated with the indicated Parpi. D, DSB repair kinetics in V3 (DNA-PKcsm) cells incubated with the indicated Parpi. Data above represent the mean ± SD calculated from four determinations in two independent experiments. E, At low IR doses, BMN673 enhances DSB end resection, increases Rad51 foci formation and possibly futile HRR. Resected ends exclude cNHEJ and promote error-prone altEJ, causing translocations and radiosensitization. F, At high IR doses, BMN673 suppresses both cNHEJ and altEJ. Resection and HRR are reduced after exposure to high IR doses (45, 46).
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